Method for quantifying and/or detecting human male DNA

ABSTRACT

According to a first aspect of the present invention, a method is provided for detecting and/or quantifying male genomic DNA in a sample, wherein the method comprises the step of amplification of a multicopy locus within the human Y-chromosome (MCL-Y), wherein said locus shares at least 80% sequence identity to a sequence according to SEQ ID NO. 3 over a stretch of at least 60 base pairs (bp). A second aspect of the present invention relates to a primer or primer pair which hybridizes under stringent conditions to a sequence according to SEQ ID NO. 3 and/or any of 4 to 11. The invention also relates to a kit.

FIELD OF THE INVENTION

The present invention is in the field of molecular biology, diagnostics,more particularly in the field of analytical and forensic sciences. Theinvention is further in the field of nucleic acid amplification andquantification, more particularly in the field of DNA quantification.

BACKGROUND OF THE INVENTION

The determination of the quantity of DNA recovered from forensic samplesas well as other samples is a critical step in the overall DNA typingprocess, but also in the detection of DNA in various other fields ofscience. A narrow range of input DNA from 0.5 to 2 ng is often needed toproduce optimal results with for example multiplex DNA typing kits.Therefore, in order to ensure that a positive result is a positiveresult and/or a negative result is a negative result due to the absenceof DNA, quantification of DNA is of absolute importance. Furthermore,the quality of standards for forensic DNA testing laboratories requireshuman-specific DNA quantification. This is due to isolation techniquesthat can recover human DNA as well as bacterial and other exogenous DNA.A number of procedures have been developed to permit quantification ofhuman-specific DNA including start-blot techniques, liquid basedhybridization assays and real-time polymerase chain reaction (PCR).Currently, real-time PCR is the dominant technique due to its widedynamic range and ease of automation.

The modern short tandem repeat (STR) kits have become much moresensitive and can obtain good results even using low amounts of DNA.Therefore, there is a desire for a method, kit and nucleic acid regionthat allows precise and accurate quantification of human DNA even in lowconcentrated samples.

There are certain quantification and detection kits already available.One such kit is the Quantifiler Human Kit (Applied Biosystems) anotherkit is Quantifiler Duo Kit (Applied Biosystems) another kit is thePlexor HY Real-Time PCR Quantification Kit (Promega). Both theQuantifiler Duo Kit and the Plexor HY Kit target an autosomal and agonosomal (Y-chromosome) target on the genome.

However, the kits currently on the market present some drawbacks.According to LaSalle et al. (Forensic Science International: Genetics,(2011) 5: 185-193) the Quantifiler Kits are more accurate in thequantification but have a lower dynamic range as the Plexor HY. ThePlexor HY offers a higher dynamic range due to the amplification of amulticopy target, but a lower accuracy. This lower accuracy can beattributed to the multicopy target. If less than the full set of 20copies on a genome amplify, because of, for example, instability in thetarget copy number, than the ratio between the amplification betweenautosomal and gonosomal (Y) target may vary. The dynamic range of thePlexor HY kit is slightly better than that of the other kit (LaSalle etal., Forensic Science International: Genetics, (2011) 5: 185-193). In astatistical comparison, it has been demonstrated a significantdifference between the two kits (LaSalle et al., Forensic ScienceInternational: Genetics, (2011) 5: 185-193).

Another important parameter in forensics is the degradation grade of theDNA that has to be analyzed. Since the amplicon size of the QuantifilerHuman and Plexor HY vary from 62 to 133 base pairs (bp), significantdifferences might be expected when the kits are applied to degraded DNA.Also, inhibitors must be taken into account. It may well be that DNA ispresent in the reaction no result is obtained due to the presence ofinhibitory substances.

In cases of sexual assault samples, a quantification of the DNA ischallenging, due to the presence of DNA molecules from both, the femalevictim as well as the male attacker. Furthermore, in a typical sample,the amount of female DNA exceeds the amount of male DNA by severalorders of magnitude. Thus, a sensitive, male specific DNA quantificationmethod which can accurately detect and quantify male DNA even in a highbackground of female DNA, is therefore of great interest.

Reported herein is a qPCR-based DNA quantification system that is highlysensitive to detect low amounts of male DNA in a high background offemale DNA and assess in parallel the male DNA degradation and/orintegrity of the male DNA.

SUMMARY OF THE INVENTION

The invention relates to a method for detecting and/or quantifying DNA,in particular the fraction of male DNA in a sample, (i) wherein themethod comprises the step of amplification of a multicopy locus withinthe Y-chromosome (MCL-Y), wherein said locus shares at least 85%sequence identity to a sequence according to SEQ ID NO. 3 over a stretchof at least 60 base pairs (bp) or with the reverse complement thereofor, (ii) wherein the locus is amplifiable with a primer pair accordingto SEQ ID NO. 1 and 2 or the reverse complement thereof.

A second aspect of the present invention relates to a primer or primerpair wherein at least one primer hybridizes under stringent conditionsto a sequence according to SEQ ID NO 3. Preferably, both primers of theprimer pair hybridize.

A third aspect of the present invention relates to a kit for performinga method according to any of the claims 1 to 10, wherein said kitcomprises: a. at least one primer selected from the group consisting of:SEQ ID NO 1 and SEQ ID NO 2; b. reagents to perform the amplificationreaction; c. instructions for performing the method according to any ofthe claims 1 to 10.

Maternal blood stream contains low amounts of cell-free fetal DNA(cffDNA) which is freely circulating. Analysis of cffDNA provides amethod of non-invasive prenatal diagnosis, testing and can be used e.g.for early determination of fetal sex. The present method also enablesthe detection and analysis of male cell-free fetal DNA and aids inidentification of fetal sex. Furthermore the present methodsignificantly decreases the risk of false sex determination of theembryo since contaminating male genomic DNA, e.g. introduced into thesample from the environment, can be assessed by the Degradation Indexgenerated by the small and large PCR system for the male targets used inthe invention.

DESCRIPTION OF THE INVENTION

In case of forensic samples from sexual assaults the female DNA usuallyexceeds the amount of male DNA. To choose the proper method for geneticanalysis it is advisable to test for male DNA present in the sample,which was collected from a crime scene and to quantify the amount ofmale DNA in order to know how much of this DNA should be used in thegenetic analysis e.g. STR reaction. The typical STR kit detects geneticlength polymorphisms on different autosomal chromosomes, but in somecases, such as with sexual assault samples, the analysis of lengthpolymorphisms exclusively on the Y-chromosome could be advantageous,because the female DNA does not contain these length polymorphisms.

Surprisingly, the inventors have found that multicopy loci on theY-chromosome are superior to other loci when used for detection and/orquantification of nucleic acids, because the sensitivity of the reactioncan be enhanced. This is an important aspect of the present inventiondue to its relevancy in the field of forensic science.

In particular, the inventors have astonishingly found that sequenceidentified in SEQ ID NO. 3 and/or sequences that share sequencesimilarity, i.e. SEQ ID NO. 4 with it may be found many times on theY-chromosome. In particular, said sequence SEQ ID NO. 3 or sequencesvery similar thereto are present nine times on the human Y-chromosome.This finding provides a valuable advantage of the present method.

The invention relates to a method for detecting and/or quantifying DNAin particular the fraction of male DNA in a sample, (i) wherein themethod comprises the step of amplification of a multicopy locus withinthe Y-chromosome (MCL-Y), wherein said locus shares at least 85%sequence identity to a sequence according to SEQ ID NO. 3 over a stretchof at least 60 base pairs (bp) or with the reverse complement thereofor, (ii) wherein the locus is amplifiable with a primer pair accordingto SEQ ID NO. 1 and 2 or the reverse complement thereof.

In the context of the present invention, the term “amplifiable” refersto the property of a locus of being amplified by any amplificationmethod. A person skilled in the art knows that the achievement of theamplification reaction depends on the experimental condition used.

The invention relates to a method, wherein the amplification step isperformed using at least one primer selected from one of the groupsconsisting of (i) SEQ ID NO. 1 and SEQ ID NO. 2, (ii) a reversecomplement of SEQ ID NO. 1 and SEQ ID NO. 2 and (iii) a primer thatshares at least 90% sequence identity with one of the primers with SEQID NO. 1 and SEQ ID NO. 2 or a reverse complement thereof.

SEQ ID NO. 1 5′GAAAGGCCTCATCAGGGCTCAG 3′ Downstream primer 81 bpfragment SEQ ID NO. 2 5′TCCTCACTGGGAAACATGAGGAATGAC 3′ Upstream primer

The invention relates to a method, wherein the amplification step isperformed using a primer pair selected from one of the groups consistingof (i) SEQ ID NO. 1 and SEQ ID NO. 2, (ii) a reverse complement of SEQID NO. 1 and SEQ ID NO. 2 and (iii) a primer that shares at least 90%sequence identity with one of the primers with SEQ ID NO. 1 and SEQ IDNO. 2 or a reverse complement thereof.

The sequences distributed throughout the genome are not all exactlyidentical. It is important that the selected primers bind also to thenearly identical sequences. Thus, ideally the locus shares at least 60%,70%, 80%, 90% or even 95% or 98% sequence identity to a sequenceaccording to SEQ ID NO. 3 over a stretch of 60 bp.

The locus may also be chosen from any of SEQ ID NO. 3 to 11. Thus, ifSEQ ID NO. 3 is claimed herein the same applies to 4 to 11.

SEQ ID NO. 3 5′GAAAGGCCTCATCAGGGCTCAGaaaggtgacccaagca (chrY:9529589-gctgggaacacacgggGTCATTCCTCATGTTTCCCAGTGA 9529669 81 bp) GGA 3′SEQ ID NO. 4 5′GAAAGGCCTCATCAGGGCTCAGaaaggtgacccaagca (chrY:9509276-gctgggaacacacgggGTCATTCCTCATGTTTCCCAGTGA 9509356 81 bp) GGA 3′SEQ ID NO. 5 5′GAAAGGCCTCATCAGGGCTCAGaaaggtgacccaagca (chrY:9488994-gctgggaacacatgggGTCATTCCTCATGTTTCCCAGTGA 9489074 81 bp) GGA 3′SEQ ID NO. 6 5′GAAAGGCCTCATCAGGGCTCAGaaaggtgacccaagca (chrY:9468664-gctgggaacacacgggGTCATTCCTCATGTTTCCCAGTGA 9468744 81 bp) GGA 3′SEQ ID NO. 7 5′GAAAGGCCTCATCAGGGCTCAGaaaggtgacccaagca (chrY:9400130-gctgggaacacacgggGTCATTCCTCATGTTTCCCAGTGA 9400210 81 bp) GGA 3′SEQ ID NO. 8 5′GAAAGGCCTCATCAGGGCTCAGaaaggtgacccaagca (chrY:9379784-gctgggaacacacgggGTCATTCCTCATGTTTCCCAGTGA 9379864 81 bp) GGA 3′SEQ ID NO. 9 5′GAAAGGCCTCATCAGGGCTCAGaaaggtgacccaagca (chrY:9359506-gctgggaacacacgggGTCATTCCTCATGTTTCCCAGTGA 9359586 81 bp) GGA 3′SEQ ID NO. 10 5′GAAAGGCCTCATCAGGGCTCAGaaaggtgacccaagca (chrY:9339191-gctgggaacacatgggGTCATTCCTCATGTTTCCCAGTGA 9339271 81 bp) GGA 3′SEQ ID NO. 11 5′GAAAGGCCTCATCAGGGCTCAGaaaggtgacccaagca (chrY:6247926-gctgggaacacacgggGTCATTCCTCATGTTTCCCAGTGA 6248006 81 bp) GGA 3′SEQ ID NO. 12 5′ ggtgacccaagcagctgggaacaca 3′ Probe for 81 bp fragmentSEQ ID NO. 13 5′ CATGAACGTCCTGGATTCTGTCACTC 3′ Downstream primer no. 3SEQ ID NO. 14 5′ TCACTCTCTGTCTTCCTCTCAAGGAATTTCTAC 3′ Downstream primerno. 4 SEQ ID NO. 15 5′ GCCATGAACGTCCTGGATTCTGTCAC 3′ Downstream primerno. 5 SEQ ID NO. 16 5′ CAGGCTCCCTGAATAGGCAGGTGTG 3′ Probe for largerfragment SEQ ID NO: 17 5′ TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc(Upstream primer ccagctgcttgggtcacctttctgagccctgatgaggcctSEQ ID NO. 2 and ttcccgattgagtcccctgacagatcctatgtaaggacctDownstream primer gtggcgcaatcccctgcaatactacaagaggatgaagccaSEQ ID NO. 13- cctgaagagggaacagagacgtcaggtgagccgttagttgLocus:chrY:9529589 + gcactggagctgtttgatgcccagtataagggggttgaca 9529947)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatgtagaaattccttgagaggaagacagaGAGTGACAGAATCCAGGACGTTCAT G 3′ SEQ ID NO. 185′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 13cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9509276 +gcactggagctgtttgatgcccagtataagggggttgaca 9509634)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatgtagaaattccttgagaggaagacagaGAGTGACAGAATCCAGGACGTTCAT G 3′ SEQ ID NO. 195′TCCTCACTGGGAAACATGAGGAATGACcccatgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 13cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9488994 +gcactggagctgtttgatgcccagtataagggggttgaca 9489352)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatgtagaaattccttgagaggaagacagaGAGTGACAGAATCCAGGACGTTCAT G 3′ SEQ ID NO. 205′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 13cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9468664 +gcactggagctgtttgatgcgcagtataagggggttgaca 9469020)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaactgaggctcctttcgtacatgtagaaattccttgagaggaagacaGAGTGACAGAATCCAGGACGTTCATG 3′ SEQ ID NO. 215′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 13cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9400130 +gcactggagctgtttgatgcccagtataagggggttgaca 9400488)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatgtagaaattccttgagaggaagacagaGAGTGACAGAATCCAGGACGTTCAT G 3′ SEQ ID NO. 225′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 13cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9379784 +gcactggagctgtttgatgcccagtataagggggttgaca 9380142)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatgtagaaattccttgagaggaagacagaGAGTGACAGAATCCAGGACGTTCAT G 3′ SEQ ID NO. 235′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 13cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9359506 +gcactggagctgtttgatgcccagtataagggggttgaca 9359864cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatgtagaaattccttgagaggaagacagaGAGTGACAGAATCCAGGACGTTCAT G 3′ SEQ ID NO. 245′TCCTCACTGGGAAACATGAGGAATGACcccatgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 13cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9339191 +gcactggagctgtttgatgcccagtataagggggttgaca 9339549)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatgtagaaattccttgagaggaagacagaGAGTGACAGAATCCAGGACGTTCAT G 3′ SEQ ID NO. 255′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 13cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:6247926 +gcactggagctgtttgatgcccagtataagggggttgaca 6248284)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatgtagaaattccttgagaggaagacagaGAGTGACAGAATCCAGGACGTTCAT G 3′ SEQ ID NO. 265′TCCTCACTGGGAAACATGAGGAATGACcccatgtgttc (Upstream primerccagctgcttgggtcaccattctgagtcctgatgaggcct SEQ ID NO. 2 andttcccgatggattcccctgacagatcctatgtaaggacct Downstream primergtggtgcaatcccctgcaatcctacaagaggatgaagcca SEQ ID NO. 14cctgaagagggaacagagatttcaggtgagctgttcagtt Locus:ggaactgaagctttttgatccccaggataaggaggttgac chrY:9549757 +acacctgcctattcagggagcctggaggctcatttcagaa 9550096)atgtagaaattgagcctcctttcatacatGTAGAAATTCC TTGAGAGGAAGACAGAGtGTGA 3′SEQ ID NO. 27 5′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 14cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9529589 +gcactggagctgtttgatgcccagtataagggggttgaca 9529927cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatGTAGAAATTCCT TGAGAGGAAGACAGAGAGTGA 3′SEQ ID NO. 28 5′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 14cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9509276 +gcactggagctgtttgatgcccagtataagggggttgaca 9509614)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatGTAGAAATTCCT TGAGAGGAAGACAGAGAGTGA 3′SEQ ID NO. 29 5′TCCTCACTGGGAAACATGAGGAATGACcccatgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 14cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9488994 +gcactggagctgtttgatgcccagtataagggggttgaca 9489332)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatGTAGAAATTCCT TGAGAGGAAGACAGAGAGTGA 3′SEQ ID NO. 30 5′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 14cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9468664 +gcactggagctgtttgatgcgcagtataagggggttgaca 9469002)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaactgaggctcctttcgtacatGTAGAAATTCCT TGAGAGGAAGACAGAGtGacA 3′SEQ ID NO. 31 5′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 14cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9400130 +gcactggagctgtttgatgcccagtataagggggttgaca 9400468)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatGTAGAAATTCCT TGAGAGGAAGACAGAGAGTGA 3′SEQ ID NO. 32 5′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 14cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9379784 +gcactggagctgtttgatgcccagtataagggggttgaca 9380122)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatGTAGAAATTCCT TGAGAGGAAGACAGAGAGTGA 3′SEQ ID NO. 33 5′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 14cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9359506 +gcactggagctgtttgatgcccagtataagggggttgaca 9359844)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatGTAGAAATTCCT TGAGAGGAAGACAGAGAGTGA 3′SEQ ID NO. 34 5′TCCTCACTGGGAAACATGAGGAATGACcccatgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 14cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9339191 +gcactggagctgtttgatgcccagtataagggggttgaca 9339529)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatGTAGAAATTCCT TGAGAGGAAGACAGAGAGTGA 3′SEQ ID NO. 35 5′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 14cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:6247926 +gcactggagctgtttgatgcccagtataagggggttgaca 6248264)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatGTAGAAATTCCT TGAGAGGAAGACAGAGAGTGA 3′SEQ ID NO. 36 5′TCCTCACTGGGAAACATGAGGAATGACcccatgtgttc (Upstream primerccagctgcttgggtcaccattctgagtcctgatgaggcct SEQ ID NO. 2 andttcccgatggattcccctgacagatcctatgtaaggacct Downstream primergtggtgcaatcccctgcaatcctacaagaggatgaagcca SEQ ID NO. 15cctgaagagggaacagagatttcaggtgagctgttcagtt Locus:chrY:9549757 +ggaactgaagctttttgatccccaggataaggaggttgac 9550118)acacctgcctattcagggagcctggaggctcatttcagaaatgtagaaattgagcctcctttcatacatgtagaaattccttgagaggaagacagagtGTGACAGAATCCAGGACaTTCA TGGC 3′ SEQ ID NO. 375′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 15cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9529589 +gcactggagctgtttgatgcccagtataagggggttgaca 9529949)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatgtagaaattccttgagaggaagacagagaGTGACAGAATCCAGGACGTTCAT GGC 3′ SEQ ID NO. 385′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 15cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9509276 +gcactggagctgtttgatgcccagtataagggggttgaca 9509636)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatgtagaaattccttgagaggaagacagagaGTGACAGAATCCAGGACGTTCAT GGC 3′ SEQ ID NO. 395′TCCTCACTGGGAAACATGAGGAATGACcccatgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 15cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9488994 +gcactggagctgtttgatgcccagtataagggggttgaca 9489354)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatgtagaaattccttgagaggaagacagagaGTGACAGAATCCAGGACGTTCAT GGC 3′ SEQ ID NO. 405′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 15cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9468664 +gcactggagctgtttgatgcgcagtataagggggttgaca 9469022)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaactgaggctcctttcgtacatgtagaaattccttgagaggaagacagaGTGACAGAATCCAGGACGTTCATGG C 3′ SEQ ID NO. 415′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 15cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9400130 +gcactggagctgtttgatgcccagtataagggggttgaca 9400490)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatgtagaaattccttgagaggaagacagagaGTGACAGAATCCAGGACGTTCAT GGC 3′ SEQ ID NO. 425′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 15cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9379784 +gcactggagctgtttgatgcccagtataagggggttgaca 9380144)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatgtagaaattccttgagaggaagacagagaGTGACAGAATCCAGGACGTTCAT GGC 3′ SEQ ID NO. 435′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 15cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9359506 +gcactggagctgtttgatgcccagtataagggggttgaca 9359866)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatgtagaaattccttgagaggaagacagagaGTGACAGAATCCAGGACGTTCAT GGC 3′ SEQ ID NO. 445′TCCTCACTGGGAAACATGAGGAATGACcccatgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 15cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:9339191 +gcactggagctgtttgatgcccagtataagggggttgaca 9339551)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatgtagaaattccttgagaggaagacagagaGTGACAGAATCCAGGACGTTCAT GGC 3′ SEQ ID NO. 455′TCCTCACTGGGAAACATGAGGAATGACcccgtgtgttc (Upstream primerccagctgcttgggtcacctttctgagccctgatgaggcct SEQ ID NO. 2 andttcccgattgagtcccctgacagatcctatgtaaggacct Downstream primergtggcgcaatcccctgcaatactacaagaggatgaagcca SEQ ID NO. 15cctgaagagggaacagagacgtcaggtgagccgttagttg Locus:chrY:6247926 +gcactggagctgtttgatgcccagtataagggggttgaca 6248286)cacctgcctattcagggagcctgggtgctcatttcagaaatgtagaaattgaggctcctttcgtacatgtagaaattccttgagaggaagacagagaGTGACAGAATCCAGGACGTTCAT GGC 3′

The inventors have astonishingly developed a very versatile system fordegradation analysis.

The degradation status/integrity of male DNA can be assessed by usingfor example, at least two differently sized genomic regions in a qPCR inone vessel. The amplified targets have to have equal amplificationefficiencies, causing co-amplification of the targets with the sameefficiency. In case of degraded male DNA the mean length of the male DNAfragments in the sample will decrease leading to a loss of efficiency inamplification of the longer PCR systems. The shorter the fragments inthe degraded male DNA sample the higher the differences in amplificationefficiencies between the shorter and larger PCR systems will become.Hereby, the integrity of male DNA or degradation status of the male DNAcan be expressed by a ratio of the quantification of the systems used.The ratio is designated as degradation index. Therefore, in one aspectof the present invention, the status of DNA integrity and/or degradationis expressed by the ratio of the quantification of the at least twooverlapping regions within the at least one locus.

Therefore, in one aspect of the present invention, the status of DNAintegrity and/or degradation is expressed by the ratio of thequantification of the at least two overlapping regions within the atleast one locus.

Here, the smaller fragment is combined with different larger fragments,depending on the extent of degradation of the nucleic acid.

The larger fragment may be a:

-   -   (i) 359 bp or 357 bp fragment (primers SEQ ID NO. 2 and SEQ ID        NO. 13),    -   (ii) a 340 bp fragment or a 339 bp fragment (e.g. primers SEQ ID        NO. 2 and SEQ ID NO. 14), or a    -   (iii) 359 bp fragment, 361 bp fragment or a 362 bp fragment (SEQ        ID NO. 2 and SEQ ID NO. 15)

Uniquely the system is set-up so that one of the primers in the primerpairs is common to the two or more fragments, being amplified. Thus, thetwo fragments small and large may have a common up-stream or down-streamprimer. Here, it is preferred that the upstream primer (SEQ ID NO. 2) iscommon to all amplifications.

The determination of percent identity between two sequences isaccomplished using the mathematical algorithm of Karlin and Altschul(Proc. Natl. Acad. Sci. USA (1993) 90: 5873-5877). Such an algorithm isthe basis of the BLASTN and BLASTP programs of Altschul et al. (J. Mol.Biol. (1990) 215: 403-410). BLAST nucleotide searches are performed withthe BLASTN program, score=100, word length=12, to obtain nucleotidesequences homologous SEQ ID NO. 1. To obtain gapped alignments forcomparative purposes, Gapped BLAST is utilized as described by Altschulet al. (Nucleic Acids Res. (1997) 25: 3389-3402). When utilizing BLASTand Gapped BLAST programs, the default parameters of the respectiveprograms are used.

The forensic workflow of sexual assault samples suggests thequantification of the male DNA before the STR reaction is carried out.This is done first to help in the decision of which kind of STR Kit hasto be used for the genetic analysis, and then to determine how much DNAwas obtained from a sample, e.g. collected from a crime scene, and howmuch of this DNA should be used in a STR reaction. Different STR Kitsare available, the typical STR Kit detects genetic length polymorphismson different autosomal chromosomes, but in some cases, such as withsexual assault samples, the analysis of length polymorphisms exclusivelyon the Y-chromosome could be advantageous, because the female DNAdoesn't have a Y chromosome.

The typical STR reaction works optimally in certain range of templateDNA and the whole analysis is very labour-intensive, thereforemethodologies are needed that ensure a very high success rate in the STRanalysis. Therefore, it is a real advantage if the quantification kitenables the user not only to surely identify the amount of DNA presentbut also to assess the absence of inhibitors, which could compromise theSTR reaction result, which would result in failure or loss of valuablesample material, which could be further purified in case criticalinhibition is observed.

According to one embodiment of the present invention, said multicopylocus within the human Y-chromosome (MCL-Y) is about 81 bp in length. Asused herein, the term “about” refers to a range comprising +/−20% of thevalue of reference. Thus, said multicopy locus can have a length rangingfrom 65 to 95 bp.

Preferably, the method comprises the step of amplification of amulticopy locus within the Y-chromosome (MCL-Y), wherein said locusshares at least 85%, 90%, 95% or 99% sequence identity to a sequenceaccording to SEQ ID NO. 3 over a stretch of at least 60 base pairs (bp)or with the reverse complement thereof.

The present method shows an improved sensibility over other commerciallyavailable methods. In particular, according to another embodiment thenucleic acid of a genome in a sample is detected and/or quantified atthe lowest concentration of 0.125 pg/μl.

According to another embodiment of the present application, theamplification product of at least one nucleic acid is between 60 and 200bp in length.

Preferably, the amplification step is performed using at least oneprimer selected from one of the groups consisting of (i) SEQ ID NO. 1and SEQ ID NO. 2, (ii) a reverse complement of SEQ ID NO. 1 and SEQ IDNO. 2 and (iii) a primer that shares at least 90% sequence identity withone of the primers with SEQ ID NO. 1 and SEQ ID NO. 2 or a reversecomplement thereof. These may be combined with a second overlappingamplicon wherein the primer pairs used have a sequence according to (iv)SEQ ID NO. 2 and SEQ ID NO. 13, SEQ ID NO. 2 and SEQ ID NO. 14, (v) andSEQ ID NO. 2 and SEQ ID NO. 15; see also FIG. 2. When measuringadditionally degradation, the amplicon SEQ ID NO. 1 and 2 is combinedwith an amplicon selected from (i) SEQ ID NO. 2 and SEQ ID NO. 13, (ii)SEQ ID NO. 2 and SEQ ID NO. 14, or (iii) SEQ ID NO. 2 and SEQ ID NO. 15.

In a preferred embodiment the amplification reaction comprisesamplifying at least two overlapping regions using at least one commonprimer, or two common primers or three common primer or more.

Preferably, the amplification step is performed using a primer pairselected from one of the groups consisting of (i) SEQ ID NO. 1 and SEQID NO. 2, (ii) a reverse complement of SEQ ID NO. 1 and SEQ ID NO. 2,and (iii) a primer that shares at least 90% sequence identity with oneof the primers with SEQ ID NO. 1 and SEQ ID NO. 2 or a reversecomplement thereof optionally combined with one or more of the pairsselected from (i) SEQ ID NO. 2 and SEQ ID NO. 13, (ii) SEQ ID NO. 2 andSEQ ID NO. 14, or (iii) SEQ ID NO. 2 and SEQ ID NO. 15.

Ideally, the amplification is performed using a primer pair with asequence according to SEQ ID NO. 1 and SEQ ID NO. 2.

Preferably, the sample originates from one of the following tissuestypes comprising whole blood, blood fractions, oral specimen, urine,human bioptic tissue or other parts of the human body upon availabilityfor isolation of a genome.

The best use of the sample is, e.g. a rape case wherein, said samplecomprises male and female genomic DNA. The ratios of the amounts may be(male/female): 1/2, 1/3, 1/4, 1/10, 1/20, 1/50, 1/100, 1/1000, 1/5000,1/10.000 or even 1/40.000, or even 1/400.000. It may also be(female/male): 1/2, 1/3, 1/4, 1/10, 1/20, 1/50, 1/100, 1/1000, 1/5000,1/10.000 or even 1/40.000, or even 1/400.000. The method has shown to beable to identify 1 pg of male DNA in 400 ng of female DNA.

The present method also enables the detection and analysis of thedegradation status of male DNA in non-degraded or also degraded femaleDNA.

Preferably, the amplification method is a polymerase chain reaction(PCR) or a real-time PCR reaction and the amount of nucleic aciddetermined is quantified either during the amplification process or asan end point measurement at the end of the amplification reaction.

At best the amplification reaction for carrying out the method of thepresent invention comprises:

-   -   a. Tris-HCl at a pH of between 8 and 8.8 and/or,    -   b. potassium salt selected from the group of, potassium chloride        and potassium sulphate and/or,    -   c. an ammonium salt, preferably ammonium chloride or ammonium        sulphate and/or,    -   d. magnesium chloride and/or,    -   e. a hot-start polymerase.

The invention relates to an oligonucleotide primer or primer pair,wherein at least one primer of said primer pair hybridizes understringent conditions to a nucleic acid with a sequence according to SEQID NO. 3 to SEQ ID NO. 11 and/or 17 to 25. Preferably, both primershybridize under stringent conditions.

As used herein, the term “stringent conditions” refer to conditionsunder which a nucleic acid having complementarity to a target sequencepredominantly hybridizes with the target sequence and substantially doesnot hybridize to non-target sequences. Stringent conditions aregenerally sequence-dependent, and vary depending on a number ofparameters, such as temperature, ionic strength and the presence ofother compounds such as organic solvents, under which nucleic acidhybridizations are conducted. The person skilled in the art is familiarwith such conditions, and thus they are not given here. Non-limitingexamples of stringent conditions are described in detail in Tijssen(1993), Laboratory techniques in biochemistry and molecularbiology—Hybridization with nucleic acid probes Part 1, second chapter“Overview of principles of hybridization and the strategy of nucleicacid probe assay”, Elsevier, N.Y.

The invention also encompasses an oligonucleotide primer or primer pairfor use in a method for detecting and/or quantifying DNA, in particularthe fraction of male DNA, in a sample according to the first aspect ofthe present invention, wherein at least one primer, preferably bothprimers, of said primer pair hybridizes under stringent conditions to anucleic acid with a sequence according to SEQ ID NO. 3 to SEQ ID NO. 11and/or 17 to 25.

Claimed are the oligonucleotides with the following sequences SEQ ID NO.1, 2, 12, 13, 14, 15 and 16 or oligonucleotides that share a sequenceidentity of no less than, 90%, 95% or 99% to these, or the reversecomplement thereof.

According to another embodiment, claimed are oligonucleotides with thefollowing sequences SEQ ID NO. 1, 2, 12, 13, 14, 15 and 16 oroligonucleotides that share a sequence identity of no less than, 90%,95% or 99% to these, or the reverse complement thereof for use in amethod for detecting and/or quantifying DNA, in particular the fractionof male DNA, in a sample according to the first aspect of the presentinvention.

Claimed is also a kit for performing a method according to any of theclaims 1 to 10, wherein said kit comprises at least one oligonucleotideprimer selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO.2, or a primer according to claim 9.

According to another embodiment, the sample subjected to the presentmethod originates from one of the following specimens comprising wholeblood, blood fractions, oral fluids, body fluids, human bioptic tissueor other parts of the human body upon availability for isolation of agenome. As used herein the terms “oral fluids” and “body fluids” refersto fluids that are excreted or secreted from the buccal cavity and fromthe body, respectively, from which a genome can be isolated. As anon-limiting example, oral and body fluids may comprise saliva, sputum,swab, urine.

In a preferred embodiment, the DNA or RNA analyzed is fragmented. Inanother embodiment, the DNA or RNA analyzed is in a composition withinhibitors.

As reported above, a typical forensic sample comprise a mixture of maleand female DNA wherein the amount of female DNA exceeds the amount ofmale DNA by several orders of magnitude. Thus, according to anotherembodiment, the sample comprises one or more additional nucleic acidsoriginating from a different genome. As used herein, the term “differentgenome” refers to genome isolated from a different subject, generallyidentified as female DNA.

According to another embodiment of the present invention, theamplification method is a polymerase chain reaction (PCR) or a real-timePCR reaction and the amount of nucleic acid determined is quantifiedeither during the amplification process or as an end point measurementat the end of the amplification reaction.

The amplification reaction according to the present method may be eithera non-isothermal method or an isothermal method.

The non-isothermal amplification method may be selected from the groupof polymerase chain reaction (PCR) (Saiki et al. Science (1985) 230:1350-1354), quantitative real-time PCR (rtPCR), ligase chain reaction(LCR) (Landegren et al. Science (1988) 241: 1077-1080). Polymerase chainreaction amplification is preferred.

The isothermal amplification method may be selected from the group ofhelicase-dependent amplification (HDA) (Vincent et al. EMBO Rep (2004) 5(8): 795-800), thermostable HDA (tHDA) (An et al. J. Biol. Chem. (2005)280 (32): 28952-28958), strand displacement amplification (SDA) (Walkeret al. Nucleic Acids Res. (1992) 20 (7): 1691-1696), multipledisplacement amplification (MDA) (Dean et al. Proc. Natl. Acad. Sci. USA(2002) 99 (8): 5261-5266), rolling-circle amplification (RCA) (Liu etal. J. Am. Chem. Soc. (1996) 118: 1587-1594), restriction aided RCA(Wang et al. Genome Res (2004) 14: 2357-2366), single primer isothermalamplification (SPIA) (Dafforn et al. Biotechniques (2004), 37 (5):854-857), transcription mediated amplification (TMA) (Vuorinen et al. J.Clin. Microbiol. (1995) 33: 1856-1859), nicking enzyme amplificationreaction (NEAR) (Maples et al. US2009017453), exponential amplificationreaction (EXPAR) (Van Ness et al. Proc. Natl. Acad. Sci. USA (2003) 100(8): 4504-4509), loop mediated isothermal amplification (LAMP) (Notomiet al. Nucleic Acids Res. (2000) 28 (12): e63), recombinase polymeraseamplification (RPA) (Piepenburg et al. PloS Biol. (2006) 4 (7):1115-1120), nucleic acid sequence based amplification (NASBA) (Kievitset al. J. Virol. Methods (1991) 35: 273-286), smart-amplificationprocess (SMAP) (Mitani et al. Nat. Methods (2007) 4 (3): 257-262).

By “isothermal amplification reaction” in context of the presentinvention it is meant that the temperature does not significantly changeduring the reaction. In a preferred embodiment, the temperature of theisothermal amplification reaction does not deviate by more than 10° C.,preferably by not more than 5° C., even more preferably not more than 2°C. during the main enzymatic reaction step where amplification takesplace.

Depending on the method of isothermal amplification of nucleic acidsdifferent enzymes are required for the amplification reaction. Knownisothermal methods for amplification of nucleic acids are the abovementioned, wherein the at least one mesophilic enzyme for amplifyingnucleic acids under isothermal conditions is selected from the groupconsisting of helicase, mesophilic polymerases, mesophilic polymeraseshaving strand displacement activity, nicking enzymes, recombinationproteins, ligases, glycosylases and/or nucleases.

The amplification methods will comprise buffers, dNTPs or NTPs inaddition to the enzymes required.

As used herein, the term “dNTP” refers to deoxyribonucleosidetriphosphates. Non-limiting examples of such dNTPs are dATP, dGTP, dCTP,dTTP, dUTP, which may also be present in the form of labelledderivatives, for instance comprising a fluorescent label, a radioactivelabel, a biotin label. dNTPs with modified nucleotide bases are alsoencompassed, wherein the nucleotide bases are for example hypoxanthine,xanthine, 7-methylguanine, inosine, xanthinosine, 7-methylguanosine,5,6-dihydrouracil, 5-methylcytosine, pseudouridine, dihydrouridine,5-methylcytidine. Furthermore, ddNTPs of the above-described moleculesare encompassed in the present invention.

As used herein, the term “NTP” refers to ribonucleoside triphosphates.Non-limiting examples of such NTPs are ATP, GTP, CTP, TTP, UTP, whichmay also be present in the form of labelled derivatives, for instancecomprising a fluorescent label, a radioactive label, a biotin label.

According to another embodiment of the present invention, theamplification reaction comprises, (a) Tris-HCl at a pH of between 8 and8.8 (at 20° C.) and/or, (b) potassium salt selected from the group of,potassium chloride and potassium sulphate and/or, (c) an ammonium salt,preferably ammonium chloride or ammonium sulphate and/or, (d) magnesiumchloride and/or, (e) a hot-start polymerase.

Preferably, the concentration of Tris-HCl is in the range from 10 to 100mM, most preferably in the range from 20 to 70 mM, the concentration ofK⁺ is in the range from 1-25 mM, most preferred in the range from 2.5 to20 mM, the concentration of NH₄ ⁺ in range from 1 to 40 mM, mostpreferred in the range from 2.5 to 30 mM, and a concentration of Mg²⁺ of0.5 mM to 8 mM in excess to the concentration of the four dNTP's, mostpreferred a concentration of Mg²⁺ of 0.7 mM to 5 mM in excess to theconcentration of the four dNTP's, a hot-start polymerase, preferentiallya hot-start polymerase allowing a hot-start time of less than 5 min,most preferred below 2 min.

A second aspect of the present invention relates to a primer or primerpair for amplifying at least one nucleic acids comprising a multicopylocus within the human Y-chromosome (MLC-Y) selected from the groupconsisting of: 5′ GAAAGGCCTCATCAGGGCTCAG 3′ (SEQ ID NO 1) and 5′TCCTCACTGGGAAACATGAGGAATGAC 3′ (SEQ ID NO 2).

According to an embodiment of the second aspect, at least one primerhybridizes under stringent conditions to a region of the Y-chromosomerepresented by multicopy loci according to SEQ ID NO. 3 to SEQ ID NO.11.

According to a third aspect of the present invention, a kit fordetecting and/or quantifying human nucleic acids is disclosed, whereinsaid kit comprises at least a primer, that under stringent conditions,binds a sequence that shares at least 80% sequence identity to asequence according to SEQ ID NO. 3 to 11 over a stretch of 80 bp,wherein in an amplification reaction, at least one nucleic acid isamplified, the locus that is amplified is a multicopy locus within thehuman Y-chromosome (MCL-Y). Is important that the primer can bind atleast SEQ ID NO. 3, 4, 5, 6, 7, 8, 9, 10 and/or SEQ ID NO. 11 or all ofthe above.

The invention further relates to a method for obtaining a degradationindex for male DNA of at least 6 when measuring degraded DNA of 350 bplength and of at least 180 when measuring degraded male DNA of 150 bplength and concentration of 2.3 ng/μl.

Preferably in the method according to the invention, the assessment ofthe status of male DNA degradation and/or integrity of one or morenucleic acids in a sample is done in parallel with the the detection ofthe one or more nucleic acids that are quantified. Preferably, themethod addresses the status of male DNA degradation and/or integrityeven in the presence of high background female DNA. In a furtherembodiment, the method may also be used for non-invasive earlydetermination of fetal sex.

The invention relates also in particular to assessing the status of maleDNA in the sample. This is done to assess the integrity or degradationstatus of the male DNA in the sample. Thus, the invention relates alsoto the use of the primers and/or probes of FIG. 2 for this statusanalysis.

EXAMPLES

The commercially available quantification kits were set up and analyzedas described in the respective handbooks. A serial dilution of humanDNAs (isolated from human blood from anonymous donors using the QIAampInvestigator Kit) and mixtures thereof at known concentrations was usedas a template for all of the three kits.

FIG. 1 shows the superiority of the present method (InvestigatorQuantiplex Pro) compared to other methods available on the market due toits increased sensitivity.

The Investigator Quantiplex Pro method provides high accuracy toquantify all amounts of used template at their correct concentrations,especially at the lowest concentrations of 0.125 pg/μl were much moreadequately quantified compared to the Quantifiler TRIO method (based onQuantifiler TRIO Kit from Applied Biosystems), which uses a multicopytarget, and the PowerQuant method (based on PowerQuant Kit from Promega)which also uses a multicopy target. Quantifiler TRIO method (based onQuantifiler TRIO Kit from Applied Biosystems showed high fluctuationsbelow 5 pg/μl and failed to quantify male DNA concentrations below 0.5pg/μl in the presence of female DNA background. The PowerQuant methodfailed to quantify the male DNA fraction below 0.25 pg/μl in thepresence or absence of background female DNA. 2 μl of given dilutions ofthe human reference DNA were used in each reaction. DNA amounts aregiven in concentrations (pg/μL) or as total amount per reaction.

FIGURES

FIG. 1 shows the superiority of the present method (InvestigatorQuantiplex Pro) compared to other methods available on the market due toits increased sensitivity.

FIG. 2 shows a possible amplification set-up. It shows differentcombinations of primer pairs for the amplification of a multicopy locuswithin the Y-chromosome (MCL-Y).

FIG. 3 Measurement of degraded male DNA according to the invention inhumans

The invention shows no significant increase for the Ct values for thesmallest PCR system (81 bp) for compromised DNA with an average fragmentlength from 1500 bp and 500 bp. Only for 300 bp and 150 bp there is anincrease of Ct values. Surprisingly the larger PCR system (359 bp) showsalready a significant shift of Ct values when applied on fragmented DNAof 1500 bp length. Furthermore, the Ct values increase consistently onevery further tested fragment length from 500 bp, 300 bp, to 150 bp andreach their maximum at 150 bp with more than 8 Ct values compared toundegraded DNA. This allows for a precise assessment of the degradationor integrity status of male DNA. 2.3 ng/μl of male DNA was used forevery fragment size or undegraded male DNA.

FIG. 4 Degradation index generated by the invention

Shown are the degradation indices (i.e. the ratio of the amount of shortfragments vs. the amount of long fragments (male S/male L)) of theinvention. Noticeably, the method according to the invention (secondcolumn) obtains extremely high indices, in particular for the smallfragments (a value of almost 190, when 2.3 ng/μl of male DNA wastested). This indicates a high sensitivity for the detection of degradedmale DNA.

FIG. 5 Measurement of degraded male DNA in background of female DNA inhumans

Different fragmented male DNAs (each 0.76 ng/μl) have been spiked intonon degraded female DNA (32 ng/μl). The invention shows no significantincrease for the Ct values for the smallest PCR system (81 bp) forcompromised DNA with an average fragment length for 500 bp. Only for 300bp and 150 bp there is an increase of Ct values. Surprisingly the largerPCR system (359 bp) shows a significant shift of Ct values when appliedon fragmented DNA of 500 bp length. Furthermore, the Ct values increaseconsistently on every further tested fragment length from 300 bp, to 150bp and reach their maximum at 150 bp with more than 8 Ct values comparedto undegraded DNA. This allows for a precise assessment of thedegradation or integrity status of male DNA in female background DNA.

FIG. 6 Detection of male cell-free fetal DNA in cell-free DNA frompregnant women

Shown is the Degradation Index (DI) generated by applying the inventionon isolated cell free DNA from a pregnant woman. The system is able todetect low amounts of male DNA with both PCR systems for male targets,the small (81 bp) and the large one (359 bp). The small system detectsthe male cell free fetal DNA which is sized between 150-220 bp. Thelarge male PCR system performs similarly to the small PCR system only onpure male genomic DNA or on contaminating male genomic DNA (spike incontrols); on male cell free fetal DNA the performance willsignificantly drop due to the size limitation of the fragments of malecell-free fetal DNA in the cell-free DNA from pregnant women. Thisgenerates a high Degradation Index (DI) for non-contaminated cell-freeDNA from women pregnant with a male embryo and a lower degradation indexfor contaminated cell-free DNA from women pregnant with a male or femaleembryo.

The invention claimed is:
 1. A method for detecting, assessing thestatus of and/or quantifying the fraction of male DNA in a sample,wherein the method comprises amplification of a multicopy locus withinthe Y-chromosome (MCL-Y), and wherein the amplification is performedusing at least one primer selected from the group consisting of: a. SEQID NO. 1; b. SEQ ID NO. 2; c. the reverse complement of SEQ ID NO. 1; d.the reverse complement of SEQ ID NO. 2; e. a primer that shares at least90% sequence identity with SEQ ID NO. 1; f. a primer that shares atleast 90% sequence identity with SEQ ID NO. 2; g. the reverse complementof a primer that shares at least 90% sequence identity with SEQ ID NO.1; and h. the reverse complement of a primer that shares at least 90%sequence identity with SEQ ID NO.
 2. 2. The method according to claim 1,wherein the amplification is performed using a primer pair selected fromone of the groups consisting of: a. SEQ ID NO. 1 and SEQ ID NO. 2; b.the reverse complement of SEQ ID NO. 1 and the reverse complement of SEQID NO. 2; c. a primer that shares at least 90% sequence identity withSEQ ID NO. 1 and a primer that shares at least 90% sequence identitywith SEQ ID NO. 2; and d. the reverse complement of a primer that sharesat least 90% sequence identity with SEQ ID NO. 1 and the reversecomplement of a primer that shares at least 90% sequence identity withSEQ ID NO.
 2. 3. The method according to claim 1, wherein theamplification step is performed using a primer pair having a sequenceaccording to SEQ ID NO. 1 and SEQ ID NO.
 2. 4. The method according toclaim 1, wherein said sample originates from whole blood, a bloodfraction, an oral specimen, urine, human bioptic tissue or another partof a human body from which a genome is isolatable.
 5. The methodaccording to claim 1, wherein said sample comprises male and femalegenomic DNA.
 6. The method according to claim 1, wherein theamplification step is performed by a polymerase chain reaction (PCR) ora real-time PCR reaction and the amount of nucleic acid determined isquantified either during the amplification process or as an end pointmeasurement at the end of the amplification reaction.
 7. The methodaccording to claim 6, wherein the amplification reaction comprises anyone or more of: a. Tris-HCl at a pH of between 8 and 8.8; b. a potassiumsalt selected from the group of potassium chloride and potassiumsulfate; c. an ammonium salt; d. magnesium chloride; and e. a hot-startpolymerase.
 8. The method according to claim 6, wherein theamplification reaction comprises amplifying at least two overlappingregions using at least one common primer.